Antibiotic x-5108 for stimulating growth

ABSTRACT

A NEW ANTIBIOTIC, DESIGNATED AS ANTIBIOTIC X-5108, IS PRODUCED BY A NEW SPECIES OF STYREPTOMYCES. THE NEW ANTIBIOTIC IS ACTIVE AGAINST GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA AND PRODUCES GROWTH STIMULATION AND INCREASED FEED EFFICIENCY IN POLUTRY.

April 18, 1972 4 Sheets-Sheet 1 Filed Aug. 28, 1970 OON 2 mmwmzDz m O02OOON UNPN PERCENT TRANSMITTANCY 00m 00m 009 OON O03 O09 O02 OOON 00mmOOOm OOmm d u a Q J. BERGER ANTIBIOTIC X-5108 FOR-STIMULATING GROWTHApril 18, 1972 4 Sheets-Sheet 2 Filed Aug. 28, 1970 N UNr N April 18,1972 Filed Aug. 28, 1970 J. BERGER ANTIBIOTIC X-51O8 FOR 'STIMULATINGGROWTH 4 Sheets-Sheet 3 FIG. 3

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ANTIBIOTIC X-5108 FOR STIMULATING GROWTH Filed Aug. 28, 1970 4Sheets-Sheet 4 FIG. 4

United States Patent Oflice 3,657,421 Patented Apr. 18, 1972 U.S. Cl.424--l22 5 Claims ABSTRACT OF THE DISCLOSURE A new antibiotic,designated as antibiotic X-S 108, is produced by a new species ofStyreptomyces. The new antibiotic is active against gram-positive andgramnegative bacteria and produces growth stimulation and increased feedefiiciency in poultry.

DESCRIPTION OF THE INVENTION This invention relates to a new antibioticand to methods for its production by fermentation, its isolation andpurification, and its use as a growth promotant in poultry. The effectsof the new antibiotic on specific bacteria in combination with itsphysical and chemical properties differentiate it from previouslydescribed antibiotics.

The present invention includes within its scope the antibiotic X-5108 inpure form, in dilute forms, and as a crude concentrate. The novelantibiotic whether present in a crude or in a more purified form isactive against a variety of microorganisms including gram-positive andgram-negative bacteria and produces marked growth promotion in poultry.

The new antibiotic, designated hereinafter as antibiotic X-S 108, isproduced by a new specie of Streptomyces, Streptomyces sp. X-5108. Thenew antibiotic-producing Streptomycete was isolated from a soil samplecollected in Bermuda. A viable culture of the organism labelled with thelaboratory designation Streptomyces sp. X-5108, subculture 31912, hasbeen deposited in the American Type Culture Collection, Rockville, Md.,where this culture has been added to the ATCC collection underRegistration No. 21386 and has been made available to the public. Thespecies of Streptomyces described herein and identified as Streptomycessp. X-S 108 includes all strains of Streptomyces which produceantibiotic X-S 108 and which cannot be definitely differentiated fromthe strain ATCC 21386 and its subcultures, including mutants andvariants. By the term mutants as used herein there is intended mutantsproduced from the described organism by various means such as chemicalmutagenic agents,

ultra-violet radiation, X-radiation, phage exposure and the like. Theproperties of antibiotic X-5108 are described herein and after theseproperties are known it is easy to dilferentiate the strains producingantibiotic X-5108 from others.

The following is a general description of the organism Streptomyces sp.X-5108, ATCC 21386, based upon characteristics such as amount of growth,pigment, morphology, etc. The descriptive colors and color chipsdesignations are generally those recommended by the InternationalStreptomyces Project (ISP): Shirling, E. B. and D. Gotttlieb, 1966,Methods for Characterization of Streptomyces Species, Intl. J.Systematic Bact. 16: 313 340. Media used to obtain the diagnosticcharacteristics and the morphological description discussed below werethose prepared of Difco Laboratories for the ISP; identification andcontent of the media are presented in Table 1.

Color names are taken from the following four sources:

ISCC-NBS U.S. Department of Commerce, 1955, The ISCC-NBS method ofdesignating colors and a dictionary of color names, National Bureau ofStandards Circular 553, U.S. Government Printing Ofiice, Washington, DC;Tresner, N. D. and E. J. Backus, 1963, System of Color Wheels forStreptomycete Taxonomy, Appl. Microb., 11: 335-338; Eckerstrom, R. andC. E. Foss, 1958, Color Harmony Manual, 4th edition, ContainerCorporation of America, Chicago, 111.; H. Prauser, 1964. Aptness andApplication of Colour Codes for Exact Description of Colours ofStreptomycetes, Z. Allg. Mikrobiologie, 4 1): -98.

Amount of growth The culture produces a well-developed and branchedsubstrate mycelium and characteristic aerial mycelium on many media. Thesubmerged growth is raised, hard and coarse, and depending on thenutrient medium employed, occurs colorless, yellow, yellow-brown orolive-brown. There are brown-black spots at the edge of the growth onyeast-malt extract agar (ISP medium 2).

Aerial mycelium and/ or en 'masse spore color Aerial mycelium ismoderately developed, with a velvety texture and pigmentedgrayish-white, light ((1) and medium-gray (2 fe), and yellowish gray (2dc)) with a thin white edge at early stage of growth. Aerial mass color(Tresner-Backus Color wheel series) is Gy on ISP medium 2 to W-gy on ISPmedia 4 and 5. Concentric rings are produced and colony sectoring occursfrequently. The color characteristics place the culture in the Grayseries (Pridham). A comparison of certain proper ties of Streptomycessp. X-S 108 with those of other members of the Gray series is presentedin Table 2.

Morphology Spore chains produced on aerial mycelium branch monopodiallyand sympodially, forming straight chains, loops, hooks, extended andirregular shot spirals (3-4 turns), also appearing in a broom-shapedarrangement. The chains are both long and short, but predominantly ofmore than 10 spores. Sclerotia are observed on most media. Spores areovalelongate and cylindrical shape (phalangiform-Tresner). The sporesurface is smooth, without any ornamentation as determined by electronmicroscopy (tomato paste agar, 10 days at 28 C.).

Physiology Soluble pigment.Traces of brown pigment are produced on yeastextract-malt extract agar (ISP medium 2) but not on ISP media 3, 4 and5.

Reverse colors.Yellow to yellow-brown plus green is produced in from 721days on ISP media 2, 4 and 5. On Dr. Prauser color guide, reverse colorsproduced in 7 days are C 004b, Co 5m, with colorless edge (medium 2),and Co 5a, Co 5b (media 4 and 5). Addition of alkali or acid produces nochange on reverse color.

Miscellaneous physiological reactions.-Culture is chromogenic (producesmelanin) on peptone-iron and tyrosine agars, as well as ontryptone-yeast extract broth. A summary of certain of the culturalcharacteristics of Streptomyces sp. X-5108 is presented in Table 3.Nitrates are not reduced in organic nitrate broth; starch is activelyhydrolyzed and gelatin is only very slightly liquified after 14 days. Anindication of other miscellaneous physiological reactions of theStreptomycete is found in Table 4. Carbon source utilization accordingto Pridham and Gottleib [J. Bact., 56, 107-114 (1948)] as follows: (11days at 28 C.): good utilization of l-arabinose, d-fructose, dmannitol,l-rhamnose, d-ratfinose and sucrose; poor uti- 3 lization of d-xylose,i-inositol and none of cellulose. Further information as to the carbonnitrogen utilization patterns of the streptomycete is found in Table 5.The culture grows well at 24 and 37 C., but not at 42 or 50.

Based on spore ornamentation, general morphology of spores and thebranching of sporophores, the colors en masse on various media andcertain biochemical and physiological reactions, it is concluded thatStreptomyces sp. X-5108 is different from any of the cultures of theGray series described in the literature.

Cultivation of the organism Streptomyces sp. X-5108 to produce thedesired antibiotic X-5108 may be carried out utilizing a variety offermentation techniques. In general, the following basic techniques canbe employed in both flask and tank procedures. In the flaskfermentation, a loopful of spores from an agar slant of the culture isinoculated into 100 ml. of nutrient medium in a 500 ml. Erlenmeyer flaskand incubated at about 28 C. on a rotary shaker for up to 7 days. Wholebroth samples are aseptically removed for in vitro assays on the 3rd,5th and 7th days. Likewise, for preparation of larger volumes of broth,inoculum is first prepared in 6 liter Erlenmeyer shakefiasks or in 5gallon Pyrex bottles, fitted for aeration, sampling etc. This broth isthen transferred to the tank fermentors. Aeration in bottles and tanksis provided by forcing sterile air through the fermenting medium. Intanks, further agitation is provided by mechanical impellers. Antifoamagents such as lard oi], soybean oil etc. are added as needed to controlfoam.

Streptomyces sp. X-5108 may be cultured in a variety of liquid culturemedia. Media which are especially useful for the production of the newantibiotic include an assimilable carbon source such as starch, glucose,molasses, and the like, an assimilable nitrogen source such as protein,protein hydrolysate, polypeptides, amino acids, corn steep liquor,ammonium salts, and inorganic cations and anions, such as potassium,sodium, calcium, magnesium, sulfate, phosphate, chloride etc. Traceelements such as cobalt, copper, iron, molybdenum, boron etc. aresupplied as impurities of other constituents of the media.

The activity of antibiotic X-5 108 can be measured in vitro by its zoneof inhibition against the gram-positive bacterium Bacillus E in theusual cup-plate agar diffusion method. Alternately the gram-positivebacterium Bacillus simplex can be employed. Both bacteria giveapproximately 20 mm. inhibition zones with an arbitrarily defined lunit/ml. solution. In this assay method, a culture of the testmicroorganism is grown in nutrient broth, for example, Trypticase SoyBroth, for 24 to 42 hours at 28 C. in a rotary shaker (100 ml. mediumper 500 ml. Erlenmeyer flask). An inoculum concentration of 0.25 to 0.5percent is used to inoculate the 4 ml. seed layer which is poured over aprehardened 20 ml. of base nutrient agar layer in a 100 ml. glass ordisposable plastic Petri dish. The plates are refrigerated for at leasttwo hours before cupping and filling with test solution containing theantibiotic. The plates are then incubated at 35 C. for 18 hours and zonediameters are measured to the nearest 0.5 mm. Calculations of unit perml. potencies are then made from standard curves.

Illustrations of the types of media that are preferably used and theantibiotic yields they support in shaken flask and in aerated tankfermentations are presented in Tables 6-9. From an examination of thedata compiled in these tables, it can be seen that complex nitrogenousmaterials from varying sources will support antibiotic production, forexample: plant materials (soybean or cottonseed flour, oatmeal, tomatopomace solids, corn fermentation solubles); animal materials (fish meal,meat meal digest, amino acid hydrolysate) and microbial cells (Torulayeast).

A number of carbon sources permit good growth and antibiotic production,for example, glucose, glycerol, dextrim and corn starch. In addition tothe inorganic salts already present in natural media, supplementationwith 4 salts such as potassium phosphate, calcium carbonate, magnesiumsulfate and trace elements will sometimes increase growth and antibioticyield (depending on the constituents already present in the basalmedium). One of the preferred media for production of antibiotic X-5108in large fermenters contains 1 percent defatted cottonseed flour, 0.5percent cornsteep liquor concentrate, 1 percent cornstarch, 0.1 percentK HPO and 0.1 percent calcium carbonate.

Streptomyces sp. X-5108 will also grow well and produce antibiotic onsome chemically defined synthetic media containing ammonium salts,nitrate or amino acids (glutamate, arginine, glycine) as nitrogensources, glucose, dextrin, starch, citrate, acetate and the like ascarbon sources, supplemented with salts such as potassium phosphate,calcium carbonate and magnesium sulphate, and with trace elementsincluding Fe++, Cu++, Mn++, C0++, Zn++. The results of fermentation ofStreptomyces sp. X-5108 on various synthetic media are shown in Table10. Generally, antibiotic yields on the synthetic media as shown inTable 10 are not as high as on complex nitrogenous media.

The production of antibiotic X-5108 is enhanced by high aeration of thefermentation medium. In addition, production of antibiotic X-5108 can beeffected at any temperature conducive to the satisfactory growth of themicroorganism. For example, Streptomyces sp. X-5l08 was grown in shakenflasks incubated at 24, 28, 30, and 32 C. Antibiotic assays after 3, 4,5 and 6 days showed that about the same maximum yield could be obtainedat any temperature from 24 to 32 C. However, the peak yield was obtainedin 3-4 days at 30 and 32 while it took 4 days at 28 and 5-6 days at 24.Ordinarily, optimum production of antibiotic X-5l08 is obtained in fromabout 2 to 10 days. The fermentation medium normally remains fairlyclose to neutral, or on the acid side, during frementation. The final pHis dependent, in part, on the buffers present, and in part on theinitial pH of the medium, which is preferably near neutral prior tosterilization.

After the fermentation is complete, a variety of procedures can beemployed for the isolation and purification of antibiotic X-5108.Suitable isolation and purification procedures include solventextraction techniques, such as batchwise extraction or counter-currentcontinuous flow liquid-liquid extraction columns, and gel permeationchromatography in a non-aqueous system.

In a preferred process, antibiotic X-5108 is recovered from the culturemedium by separation of the mycelium and any undissolved solids from thefermentation broth by conventional means such as by filtration orcentrifugation. Antibiotic X-5108 is then extracted from the filtered orcentrifuged broth using either batchwise or countercurrent distributionextraction techniques. The solvent extraction may be performed using apH range of from about 3 to about 7.5 and employing as the solvent waterimmiscible esters such as ethyl acetate, amyl acetate, butyl acetate andlike aliphatic esters; with butyl acetate being preferred. A preferredsolvent system for use with the counter-current distributionpurification technique consists of a mixture of ethyl acetate,isopropanol, and 0.1 M aqueous secondary sodium phosphate solution.

Final purification of antibiotic X-5l08 can be achieved by gelpermeation chromatograph. This purification technique is accomplished byadsorption of pre-purified preparations of the antibiotic, for example,preparations obtained by solvent extraction techniques, on cross-linkedor polymerized dextran gels. In a preferred aspect of this finalpurification technique, the pre-purified antibiotic preparation ischromatographcd on Scphadex LH-ZO eluting with alcohol.

After filtration or centrifugation of the fermentation medium, thinlayer or paper chromatography techniques can be employed to analyze forantibiotic X-S 108. Because of the color characteristics of theantibiotic, visualization of the spots can be achieved using thefluorescent indicator method; in addition, bioautography can also beemployed advantageously. The chromatography may be carried out on paperbut is preferably performed on silica gel glass plates. The solventsystem employed for the thin layer chromatograms consist of chloroform,methanol and aqueous ammonium hydroxide.

The novel antibiotic of this invention, antibiotic X- 5108, uponpurification exists as an amorphous yellow substance. Cationic salts ofthe antibiotic can be formed employing the antibiotic Xl08 and apharmaceutically acceptable inorganic or organic base. Among the saltsof antibiotic X-5108 which can be formed are the alkali metal salts,such as the sodium and potassium salts, and the alkaline earth metalsalts such as the calcium salt.

These salts can be formed for example using aqueous solutions of alkalimetal and alkaline earth metal hydroxides. Thus solutions of antibioticX-5108 in aqueous sodium hydroxide, aqueous potassium hydroxide orcalcium hydroxide form the sodium, potassium or calcium salt. Thesesalts can be used for the same biological purposes asv the antibiotic.Since solutions of salts of antibiotic X- 5108, especially the sodiumsalt, are considerably more stable than solutions of the antibiotic innon-salt form, it is preferable to use slightly alkaline buffers forpurification by counter-current distribution and ammoniacal solventmixtures for thin layer chromatography, thus minimizing theconcentration of antibiotic in solution.

The antibiotic contains the elements carbon, hydrogen, oxygen andnitrogen in substantially the following percentages by weight:

Antibiotic X-5108 Sodium salt The antibiotic X-S 108 is soluble inalcohols, for example, methanol, ethanol, 1- and 2-propanol, andtert-butyl alcohol; water-immiscible esters, for example, ethyl acetate,amyl acetate, butyl acetate and like aliphatic esters; and chloroform.The antibiotic is insoluble in water. The sodium salt of antibioticX-5108 is soluble in water, lower alcohols such as methanol, ethanol,isopropanol and butanol, and N,N-dimethylformamide; is slightly solublein amyl alcohol, tetrahydrofuran and dioxane; is very slightly solublein acetone, amyl acetate, butyl acetate and ethyl acetate; and isinsoluble in benzene, chloroform and ethyl ether.

The following are various physical characteristics of antibiotic X5108:

The optical rotation of the sodium salt of antibiotic X-5l08 is [u]=82.8 (ethanol, c.=0.52).

The infrared absorption spectrum of antibiotic X-5108 in a KBr pellet isshown in FIG. 1. The antibiotic exhibits characteristic absorption inthe infrared region of the spectrum at the following wave lengthsexpressed in reciprocal centimeters:

Broad band at 3400 Strong band at 1660 Broad band at 1580 Prominentbands at 1580, 1380, 1250, 1100, 1040 and 995.

The infrared absorption spectrum of the sodium salt of antibiotic X-5108in a KBr pellet is shown in FIG. 2. The sodium salt exhibitscharacteristic absorption in the infrared region of the spectrum at thefollowing wave lengths expressed in reciprocal centimeters:

Broad band at 3400 Prominent bands at 1645, 1570, 1500, 1388, 1105 andThe ultra-violet absorption spectrum of antibiotic 6 X-5108 at varyingpH levels is shown in FIG. 3. Ultraviolet maxima occur at:

max.

The nuclear magnetic resonance spectrum of the antibiotic X-5108 isshown in FIG. 4. The NMR spectrum was obtained using CDCl as the-solvent and tetramethyl silane (TMS) as the internal standard. The NMRspectrum exhibits prominent signals at 0.926, in the region between 1.66and 2.036, at 3.16 and 3.435 and in the olefinic region.

Antibiotic X-5108 has a broad antimicrobial spectrum as shown in Table11. The antimicrobial spectrum was determined by employing agardiffusion cup-plate assays as described earlier.

Antibiotic X-5108 exhibits low oral and parenteral toxicity, markedantistreptococcal activity both systemically (oral route) and local(subcutaneous route), activity against pneumococcus and in caecalcoccidiosis. In mice, the antibiotic is relatively atoxic orally andsubcutaneously (LD values= 2,000 and 1,320 mg./kg. respectively). Theantibiotic is active orally (CD =52 mg./ kg.) and subcutaneously (CD =44mg./ kg.) against Streptococcus pyogenes, is active against Diplococcuspneumoniae (CD =807 mg./kg. p.o. and 283 mg./kg. sbc), as well asagainst the gram-negative Proteus vulgaris infection.

Antibiotic X-5108 exhibits activity as a poultry growth promotant andbrings about enhanced feed eificiency in the animals. Thus, in a furtherembodiment of the present invention, antibiotic X-5108 is employed asthe active ingredient in new and useful compositions which upon oraladministration to poultry result in an increased growth rate and anenhanced feed efficiency in the animals. Administration of thesecompositions is accomplished through the production of nutritionallybalanced poultry feeds that satisfy the animals nutrient requirements inaddition to supplying the active growth promotant antibiotic X-5108.

When antibiotic X5108 is used in the preparation of the growth promotantcompositions, the antibiotic component is selected from the groupconsisting of the antibiotic and any pharmaceutically acceptablecationic salt thereof, preferably the sodium salt. In the discussionthat follows, the term antibiotic X-5108 will be used to denoteantibiotic X-5108 and its pharmaceutically acceptable cationic salts.

The growth stimulating compositions of this invention containing as theactive ingredient antibiotic X-5l08 are prepared by a variety ofmethods. Following one such method, the antibiotic is added directly toan edible nontoxic carrier. It is preferred that the carrier be amaterial having nutritional value for poultry; with a high energypoultry feed being the most preferred carrier. In the case where theantibiotic is added directly to the feed, the

, mixing step can be accomplished by employing known techniques. Forexample, the nutrient materials which comprise the poultry feed are fed,either individually or collectively, into a batch mixer and theantibiotic is then added. The mixer is operated until the productcontains a uniform distribution of ingredients throughout.

The nutrient materials used as poultry feeds and for the purpose of thisinvention as carriers for the antibiotic X-5108 will vary to some extentdepending upon the specific needs of the type of poultry being fed andon the final use being made of the animals. However, for the most partthese feeds will contain sources of protein, such as fish meal, soybeanmeal, corn, peanut products and the like; and sources of carbohydrates,such as grains, meals, fiours, sugars and the like. In addition, themineral and vitamin balances for the animals can be maintained by theincorporation into the feed of the required minerals, i.e., sodium,potassium, magnesium, calcium carbonate, etc. and vitamins, i.e. vitaminA, B D and thiamine. Of course, the feed may also contain otherconventional feed additives.

In a preferred method of producing the growth promoting compositions ofthe invention, the active ingredient antibiotic X-S 108 is incorporatedinto a concentrated premix which can then be added to the poultry feed.In preparing the solid form pre-mix containing antibiotic X-5108, anysuitable carrier or extender material can function as the inertingredient provided that it be inert to the active antibiotic additiveand be non-toxic to the poultry receiving the composition. Numeroussolid materials satisfy these requirements and, therefore, will functionsuccessfully for the purposes of the present invention. Representativeof such solid materials are mineral sources such as ground oystershells, edible cereals, vegetable, marine or animal materials such asare present in commercial animal feeds, corn meal, citrus meal, soybeanmeal, fish meal, meat scraps, dried fermentation residues and the like.

Antibiotic X-S 108 may be blended with one or more of the suitable solidmaterials discussed above into a mash, pellet, or any desiredconfiguration by any known and convenient technique. For example, thecomposition can be formed by finely dividing or pulverizing the activeingredient and the inert ingredients using any commercially availablegrinder. If the feed material is not present when the grinding or thepulverizing is effected, the resultant material can be distributed inaccordance with the present invention in any conveniently available feedmaterial.

The quantity of antibiotic X-5108 required to achieve the desired growthrate stimulation and feed efficiency enhancement is critical, but mayvary within the prescribed range. Preferably, when used in conjunctionwith the animals feed supply, the improved growth promoting compositionof the present invention comprises a supplemental poultry feed havingdispersed therein per 100 parts by weight of feed from about 0.0001 partby weight to about 0.01 part by weight of said active material; namely,antibiotic X-l08 or pharmaceutically acceptable salts thereof. Higherconcentrations of antibiotic X-5 108 than 0.01 part by weight per 100parts by weight of feed do not generally show improved results over theresults obtained with 0.01 part per 100 concentration. Thus, it is notadvantageous to use amounts greater than 0.01 part by weight of activeingredient per 100 parts by weight of feed. In a preferred embodiment ofthe invention, the novel growth promoting composition comprises asupplemental poultry feed containing per 100 parts by weight of feed,from about 0.0005 part by weight to about 0.0025 part by weight of theactive ingredient antibiotic X-5108.

As indicated above, the preferred practice of the invention involvesinitially preparing a concentrated premix containing the activeingredient antibiotic X-5108. Preparation of a pre-mix which can laterbe added to the feed provides a convenient method of using the growthpromoting composition and insures the proper distribution of the activeingredient throughout the feed. The amount of antibiotic X-S 108 presentin the pre-mix is not critical to the operability of the invention. Theobjectives of the invention are achieved, regardless of the level ofantibiotic X-5108 in the pre-mix, by utilizing a quantity of the pre-mixcapable of providing a final feed containing an effective level ofantibiotic X-SIOS as defined EXAMPLE 1 Fermentation of Streptomyces sp.X-5108 A spore suspension of Streptomyces sp. X-5108 from a nutrientagar test tube slant was inoculated into a 5 gallon Pyrex aerated bottlecontaining 15 liters of medium of the following composition:

Percent Soybean flour 1 Brown sugar 1 Cornsteep solids 0.25 K I-IPQ, 0.1CaCO 0.1 Lard oil as antifoam 0.5

After four days growth at 28 C., with aeration, the filamentous growthwas transferred to a gallon stainless steel fermentor containing 60gallons of medium of the following composition:

Percent Defatted cottonseed flour (Proflo) 1 Cornstarch 1 Cornsteepsolids 0.25 CaCO 0.1 K HPO 0.1

Lard oil as antifoam.

The pH of the medium was adjusted to 6.8 before inoculation. The tankwas aerated and growth allowed to proceed for 5 days. The contents ofthe fermentor were then filtered off with the aid of diatomaceous earth(Hyfio Filter Cell). The filtrate was adjusted to pH 3.5 and extractedwith one-half volume of butyl acetate. The clarified extract wasconcentrated under reduced pressure at below 40 to a brown syrup, which,after trituration with petroleum ether, yielded a solid powder,antibiotic X- 5108, assaying units Bacillus E per mg.

EXAMPLE 2 Purification of antibiotic X-5108 by batchwise solventextraction The batchwise solvent extraction process was performed on3000 gallon fermentation batches which assayed in vitro about units/ml.against B. simplex. The filtered broth was extracted with butyl acetateat pH 7.5. The organic extract, after water washes, was flashconcentrated at temperatures below 50, and to the resulting anhydroussolution was added diethyl sodiomalonate reagent to bring the pH to 9.0.The yellow precipitate that formed was filtered off and washed withbutyl acetate. It was then partitioned between water-butyl acetate andthe pH adjusted to 4. The organic phase was separated and treated asabove, except that the diethyl sodiomalonate reagent was added to pH8.5. This second precipitate was once again acidified and extracted withfresh butyl acetate. The extract was then treated with Darco G-60, andthe sodium salt of antibiotic X5108 was finally precipitated by additionof diethyl sodiomalonate to pH 8.0. The yellow sodium salt thusobtained, after washing and drying, assayed in vitro about 430 units/mg.against B. simplex. The above described extraction procedure issummarized in the following flow sheet.

Flow Sheet for Isolation of Antibiotic X-5108 3000 gals. of Whole Broth(5-0 day harvest) 1 Cells filtered ofi with aid of Hyflo 2600 gals. ofFiltered Broth (pH 7.5) [(30 hen necessary, pH adjusted with H3PO4)xtracted with 600 gals. butyl acetate 570 gels. of 1st Butyl Acetate" IWashed twice with 25 gals. of H 1 Flash concentrated 80 gals. of 1stButyl Acetate Concentrate {Added diethyl sNogliomalonate reagent to pH 9(ca. 1 to Step 1 Step 2 Step 3 1.5 liters of 1.7 Precipitate filteredoff, washed with 1 gal. of butyl acetate 1st Precipitate" [Dissolved in25 gals. of H 0, pH adjusted to 4 with 15 Step 4 percent HaPOl;Extracted twice with butyl acetate (20 10 gals.)

28 gals. of 2nd Butyl Acetate" i Washed twice with 2 gals. of H 0 1Flash concentrated 4 gals. of 2nd Butyl Acetate Concentrate (Addeddiethyl sodiomalonate reagent to pH 8.5; l Precipitate filtered oli,washed with 1 gal. of butyl acetate 2nd Precipitate" lDissolved in 20gals. of H 0, pH adjusted to 5 with 15 Step 5 Step 6 Step 7 percentH3PO4,

Step 8 Extracted twice with butyl acetate (15 7.5 gals.)

21 gals. of 3rd Butyl Acetate Washed twice with 2 gals. of H 0 Flashconcentrated 4 gals. of 3rd Butyl Acetate Concentrate Stirred with 50grams of Darco G-60 for 1 hour at room Stop 0 temperature, filtered andflash concentrated 2 gals. of "4th Butyl Acetate Added dlethylsodiomalonate reagent to pH 3.0;

Precipitate filtered off, washed with 1 gal. of butyl acetate,

then 1 gal. of pet. ether (30-60"), dried at 56 and 0.5 mm.

for 36 hours Sodium Salt of Antibiotic X-5l08 as a yellow amorphous soid Step 10 Step 11 EXAMPLE 3 Purification of antibiotic X-5108 bycountercurrcnt distribution Instrument:

Number of tubes-20O Volume of upper phase40 ml. Volume of lower phase40ml.

Charge: 5 g. of antibiotic X-5108 obtained by solvent extraction ofcrude fermentation broth, dissolved in 40 ml. upper phase.

Solvent system: Ethyl acetate, isopropanol, aqueous 0.1 M secondarysodium phosphate solution, 12:9:20 v./v.

Distribution characteristics:

Number of transfers 200 Peak tube after 200 transfers 159 Distributionratio 3.88

10 EXAMPLE 4 Purification of antibiotic X-5108 by chromatography onSephadex LH-20 An ethanolic solution of 300 mg. of a sample of theantibiotic previously purified by countercurrent distribution wasadjusted to pH 8-9 with a sodium methoxide solution (wet indicatorpaper) and applied to a Sephadex LH-20 column (290 x 41 mm),equilibrated with 3A alcohol. The column was developed with 3A alcohol,the major antibiotic zone emerging at an effluent 'volume of 950-1200ml. Following fractions contained a number of colored zones withnegligible biological activity. The active fractions were pooled andevaporated to a small volume to which ether and petroleum ether wereadded to precipitate the antibiotic. The resulting yellow solids, thesodium salt of antibiotic X-5108, exhibited only one spot upon tlc, R=0.l9 (silica gel F-254, detection by UV light) and R =0.29 (silicagel/kieselguhr, bioautographic detection).

EXAMPLE 5 Preparation of antibiotic X-5 108 from the sodium salt 0.464g. of the sodium salt of antibiotic X-5108 obtained by chromatography onSephadex LH-20 was dissolved in 4.6 ml. ice water and 10 ml. ethylacetate were added to form a 2-phase system. To this mixture, containedin a separatory funnel, was added 1 ml. of primary sodium phosphatesolution (50 g. of NaH PO-H O in ml. water). The initially formedprecipitate dissolved on shaking. The aqueous phase was discarded andthe organic phase washed four times with one volume of ice water eachand dehydrated azeotropically (ethanol). The concentrate (2 ml.) wasdiluted with two volumes of ethyl acetate and diethyl ether addeddropwise to turbidity and finally 30 ml. petroleum ether were added. Theresulting precipitate was collected by filtration and dried at roomtemperature to yield antibiotic X-5108.

EXAMPLE 6 Measurement of the growth stimulating effects of antibioticX-5108 A basal ration was prepared containing the following namedingredients in the quantities hereinafter indicated:

Percent by weight Ground yellow corn 56.075 Meat and bone meal (50%protein) 4.000 Fish meal (60% protein) 4.000 Soybean meal (50% protein)28.000 Dehydrated alfalfa meal 1.000 Animal fat 4.000 Methionine 0.200Rock phosphate 0.250 Calcium carbonate 1.200 Iodizcd salt 0.250 Vitaminsupplement 1.000 Trace mineral supplement 0.025

Antibiotic X-5 108 was added to this ration in a ratio of 50 milligramsof antibiotic per kilogram of ration.

The growth stimulating effects of antibiotic X-5108 were determined byallowing poultry to feed, ad libitum, on the antibiotic supplementedration. In the test, one day old Cornish Cross Sexed Broiler Chicks wereused. The test utilized l0 chicks per replicate (5 males and 5 females).The replicate groups were permitted access to the ration. A plannedrandom distribution of the replicates was made to equalize factors ofheating, light and position. The birds were observed over a two weekperiod, with group weight being determined several times during theperiod and individual .weights being determined at the end of 14 days.Feed consumption was also recorded and improvement in feed efficiency,as compared to the control, was calculated.

A control experiment was carried out simultaneously, in the mannerdescribed as above, except that the chicks which were used in thecontrol test were allowed to feed, ad libitum, on a ration whichcontained the same nutrient ingredients but did not contain theantibiotic X-S 108 additive.

The average gain for each test group is divided by the average gain ofthe negative control group and the quotient multiplied by 100 to yieldthe percent weight gain. Gain is the final body weight of the chick atthe end of the 2 week test minus the beginning weight of 1 day of age.

(Av. final wt.-av. initial wt. of test group) (Av. final \vt.av. initialwt. of control group X 100: percent wt. gain TABLE 12 Antibiotic Two-Percent suppleweek Feed improved ment mg.l gain, I Percent eifiteedefli- Supplement kg. feed g.=l=2SE 3 gain ciency ciency Basal control154518 100 1. 55 Antibiotic X-5108- 50 185:1:12 120 1.39 +12 l Averagefor 40 chicks. 9 Standard error of the mean.

TABLE 13 Level N 0. 'IWo- Per- Percent 1e of week cent Feed improvedSupplement mgJkg. birds gain, g. gain efl. iced e Basal Control 48 149100 1. 54 Antibiotic X-5108 50 24 175 117 1. 37 +12 Basal control 30 152100 1. 47 Antibiotic X5108- 50 18 173 114 1. 38 +7 Basal control 30 146100 1. 59 Antibiotic X-5108. 25 18 180 123 1. 37 +16 Basal control 48149 100 1. 51 Antibiotic X5108 18 183 123 1. 39 +9 Basal control 48 149100 1. 54 Antibiotic X-5108- 10 18 185 124 1. 41 +13 13 asal control 48149 100 1. 54 Antibiotic X-l08- 100 18 182 122 1. 33 +14 From theforegoing table, it is seen that the chickens fed on the rationsupplemented with 50 mg. of antibiotic X-5l08 per kilogram of feedexperienced an increased growth rate as compared to the control. At thesame time, as indicated by a percent improvement in feed efiiciency, thesame birds made more eflfective use of their feed.

EXAMPLE 7 The experiment described in Example 5, including the control,was repeated several times using the same basal ration as in Example 5.In these trials the level of antibiotic X-5108 was varied. The trialchickens were fed on supplemented feed rations containing the activeingredient in a ratio of 5, 10, 25, 50 and 100 mg. per kg. of feed.These trials were repeated to confirm the results. In the control testfour replicates of ten birds were allowed to feed on a basal rationdevoid of the antibiotic supplement. In all tests, growth rate over atwo week period was observed in comparison to the control group. Feedconsumption was also recorded and improvements in feed efiiciency incomparison to the control were calculated. Table 13 records the resultsof this series of experiments.

Distilled water--1.0 liter pH 7.0 to 7.2, before autoclaving 12 Dispense5 ml. of broth into test tubes with a diameter of 20 mm. or more.

Medium 2.Yeast extract-malt extract agar:

Bacto-Yeast Extract (Difco)4.0 g. Bacto-Malt Extract (Difco)--10.0 g.Bacto-Dextrose (Difco)4.0 g. Distilled water-1.0 liter Adjust to pH 7.3,then add Bacto Agar-20.0 g. Liquify agar by steaming at C. for 15-20minutes.

Dispense appropriate amount for slanting into at least 6 tubes for eachculture. Sterilize by autoclaving; cool tubes as slants.

Medium 3.Oatmeal agar:

Oatmeal-20 g. A'gar18.0 g.

Cook or steam 20 g. oatmeal in 1000 ml. distilled water for 20 minutes.

Filter through cheese cloth.

Add distilled water to restore volume of filtrate to 1000 ml.

Add trace salts solution-1.0 ml. solution of 0.1 g. of each of FeSO -7HO, MnCl '4H O and ZnSO -7H O in 100 ml. distilled water.

Adjust to pH 7.2 with NaOH.

Add 18 g. agar; liquify by steaming at 100 C. for 15-20 minutes.

Medium 4.-Inorganic salts-starch agar:

Solution 1: Difco soluble starch 10.0 g. Make a paste of the starch witha small amount of cold distilled water and bring to a volume of 500 ml.

Solution II:

K HPO (anhydrous basis)1.0 g. MgSO -7H Ol.0 g.

NaCl-1.0 g.

CaOO 2.0 g.

Distilled watcr500 ml.

Trace salts solution1.0 ml. of solution as in Medium 3.

pH should be between 7.0 and 7.4. Do not adjust it it is within thisrange.

Mix starch suspension and salts solution.

Add agar (Difco)20.0 g.

Liquify agar by steaming at 100 C. for 15-20 minutes.

Medium 5.Glycerol-asparagine agar:

L-Asparagine (anhydrous basis)--l.0 g. Glycerol-10.0 g. KzHPO4(anhydrous basis)l.0 g. Distilled water-1.0 liter Trace saltssolution1.0 ml. of solution as in medium 3 The pH of this solution isabout 7 .0-7.4. Do not adjust if it is Within this range. Agar200.0 g.Liquify agar by steaming at 100 C. for 15-20 minutes.

Medium 6.Peptoneyeast extract iron agar:

Bacto-Peptone Iron Agar, dehydrate (Difco)-36.0 g. Bacto-Yeast Extract(Difco)1.0 g. Distilled water-4.0 liter.

pH should be 7.0-7.2 before autoclaving; adjust if necessary.

Liquify agar by steaming at 100 C. for 15-20 minutes.

Medium 7.Tyrosine agar:

Glycerol-45.0 g.

L-Tyrosine (Difco)--0.5 g. L-Asparagine (Difco)l.0 g. K HPO (anhydrousbasis)0.5 g.

Medium 7 .Tyrosine agarCont-inued Sterilize at 15 lbs. pressure for 30minutes so .7 5 pH adjusted=6.9 NaCl0.5 g.

TABLE 2.COMPARISON OF PROPERTIES OF STREPTOMYCES X-5l08, AND OTHERMEMBERS OF THE GRAY SERIES S.sp. Rivett Streptomyces S. antibiotic us S.aureofaczem S. qnseoluteus S. purpureofuscus S. viridiand PetersProperty X X-5108 Pfizer 15784-1 Lederle A-377 Urnezawa Weksman facienrNo. 11

Spore surface Sm sm- Morphology RA. RA. Color Gy Gy. Melanin productionAntibiotic production X-5108.--

tol

Carbon source:

Glucose 1 Arabinose l-Rhamnose Galactose 1 Data on cultures other thanStreptomyces X-5108 were taken from the literature.

No'rE.Sm=smooth on electron microscope; RA =retinaeulum-apertum; Gy=Gray series; +=growth; -=no utilization.

TABLE 3.CULTURAL CHARACTERISTICS OF STREPTOMYCES SP. X-5l08 [Incubationz14 days. Temperature: 28 0.]

Amount of Soluble Medium (solid) 1 Growth Aerial mycelium and/or sporespigment Reverse color Czapeks sucrose agar Good Aer. mycwhitish spogood, white Sl. dk. br S1. yellow. Asparagine-dextrose, 0.25% "do!Aeintnye. fuzzy to powdery, white, becoming smoke gray sporu- NoneOrange to pinard a e ellow. Tomato paste-peptone agar do-.- Aer. mycwhitto It. g ay; spo good, g ay do OI ange bufi. Starch agar do. Spor lationgood, tuming smoke-gray, white edge do Orange yellow. Krainskys glucoseagar ..d- Aer. myc-Wlnt such g d, turning sm k -grey. do Lemon-yellow,Mycophil agar (BBL) -.(10 N sporfllafirm l. dk.. Sl.yellow. Yeastextract-nutrient agar d0 do i Dk Grayish to yellow- Glycerol agar. Fairto good 3 Wet, cream-colored. None SLyellow. Potato plug GoodUnsporulated i i B1 Carrot plug. .do do 1 Growth was also good on all ofthe following liquid media: tryptone broth (dk. sol. pigm; negativeindole 3 days), FDA nutrient broth Czapek sucros e broth, Krainskyglucose broth and calcium citrate-glycerol broth.

2 Growth was good in 8 days on this medium at emperatures of 24, 28, 32,and 37, but nor at 42 C.

3 10 days.

TABLE 4.-PHYSIOLO GICAL REACTIONS OF STREPTOMYCES SP. X-5108 IncubationMedium period, days Amount of growth Physiological reaction or y,nitrate broth 8 Moderate, unspor No nitrate reduetio g 13 Good; dk.pigm. Do. no gas 5 Fair; dk. sol. pigm.. No gelatin liquefaction. o 12Fair to good gr; br.-b1k. pigrm; unspor Slight liquefaction. peptommonagar (K11g1e1) 14 Good; sporulatlon v. Sligh Chromogenie (melaninproduced). I Litmus milk 3}; gggg 113E135 Elia-{eag cglor unchanged: 110curdline or clearinga Good; SL 5 spomlamn 0 or S mwn no wrdline orclearing- 10 Very good growth; mostly spornlated, lt. gray Slight dk.pigment.

3 1 0 m1 [Incubationz14 days. Temperature: 28 0.] Adjust to PH Carbonsource Utilization 1 Carbon source Utilization- Bacto-Agar20.0 g.l-Arabinose 1 3 Liquify by steaming at 100 C. for -20 minutes. jggfiggij g 3 Trace salts solution (use as directed in media 3, 4, 5, and g a7): Glucose 3 2 e' z e- 5335359.: 2 8 MnCl -4H O-0.1 g. gaflirfiosm 3zns0 -7H 0 0.1 g. *3, Distilled water-400.0 m1. g-ggl et se" g 1 110 e-Tomato paste agar (Berger): ggl l moseu g i Maltese? 3 1 Glucose 10.0 g.Dextrin 0 3 K HPO -1.0 g. l gul gzol o 3 ry ri o i. 0 d1-Trypto-phane 2a Paste SorbitoL- 1 Negative control.-." 0-1 W1lson s peptone-1.0 g.Glycerol e1 CaCO3 2'0 1 3 good utilization, 2 fair utilization, 1 poorutilization, 0 Agar-15 .0 g. no utilization. Negative control, no carbongave 0.

Tap water-1,000.0 ml.

15 Distilled water spore suspensions were used for inoculations and weretaken from tomato agar slants. The utilization of carbon and nitrogencompounds was tested on the following basal medium:

1 All media contained 0.1%11K HPO and 0.1% CaCO unless otherwisementioned. One ml. of spore suspension of Streptomypces sp. X-5108 wasinoculated into each 100 ml. medium.

KH PO -2.38 g. 5 K HPO 5 .65 g. MgS -7H O-1.00 g. CuSO -H O-0.0064 g. SOJH O 0,0011 TABLE 8.EFFECT 0F NUTRIENT VARIATIONS ON ANTI- BIOTIC YIELDBY STREPTOMYCES X-5108 IN SHAKE MnCl -4H 0-0.0079 g. FLASKS AND AERATEDKETTLES ZnSO -7H O-0.00l5 g. A [Unless otherwise indicated, e3c1l17nI12dIiIu0ni c]ontained 0.5% CaCOa and Distilled water-1 liter a 2 4Antibiotic yields, B. Medlum adlusted to PH 15 Composition of themedium, percent simplex units per ml. To test carbon sources on thisbasal medium, 2.64 g. Tomato Distil- Tanks, 0 days per liter of (NH S0was added as the common nitro- It Flasks. (duplicate em sol d solubl s th Gl gen source; the carbon sources were added at a 1% con- 1 s e s are0050 7 days runs) centration level, except for the sodium salts of theorganic 1 1 1 1 150 100.120 2 2 0 1 0. 5 100 71, 46 acids which wereused at 0.15% level. To test nitrogen 3 0 2 1 0. 5 91 88,119 sources,the synthetic agar basal plus 1% starch plus nitrog i i g 1 i 8 gensource at a concentration of 0.1 g. nitrogen per liter 1:11:: 1 1 0 54(1) was 7 1 1 4 9.1 9.1 94 97 1 Not done. 9 Proflo. 3 Starch. I CaCOz. 5KgHPOl- 6 4 days.

TABLE 6.-EFFECT OF NUTRIENT VARIATIONS 0N ANTI- BIOTIC YIELD BYSTREPTOMYCES sp. X-5108 IN SHAKE FLASKS [All media contained 1% dextrin,0.1% KzHPOi and 0.1% CBCOs'. One ml. of spore suspension of Streptomycessp. X-5108 was inoculated into each 100 ml. of medium] TABLE 0.TANKFERMENTATIONS WITHVARIO US MEDIA Experi- Age Antibiotic ment Allnitrogen sources added at 1% in potency in E Experiment Composition ofmedium in Day of Potency in number levels except 17 and 23 (2%) daysunits per ml. number I percent harvest E units/ml.

1 Meat Meal digest 6 50 K12 l distillers dried solubles l dex- 6 67 2BY-100 (Commercial Solvents 5 45 trin, 0.1 K1HPO1, 0.1 CaCO:. 3.. Proflo(Traders Oil Mi Co) 5 23 K13 1 Na-l-glutamate, 1 dextrin 0.1 5 199 4.-Oatmeal (Quaker Oats Co.). 5 32 eornsteep solids, 0.15 KzlElPOi, 5.-Soyalose (deiatted soybean do 5 30 0.05 MgSOi. 8.- Protein peanutmeal..- 5 41 K11 1 tomato pomace solids, 1 dis- 7 200 9-. P n coc n meal5 50 tillers dried solubles, 1 corn- 10. Tomato pomace Solids.- 5 57starch 0.5 glucose, 0.5 CeCOa, 11. Linseed oil meal 5 37 0.1 K2 P04. 12.Cornmeal 6 33 K4 0.5 Proflo 0.5 distillers dried 4 91 13 Corn distillersdried grain with solu- 5 74 solubles, 0.5 tomato pomace bles (H.Walker). solids, 0.5 meat extract paste 14 Soludri (Sehenley Distillers)5 51 (protopeptone No. 366), 1 15- Fishmeal (Gortons) 5 25 starch, 0.1OaCOa, 0.1 KzHPOi. 16- Fish scraps (Wilkinson) 5 16 K16 1.0 Profio 0.5cornsteep liquor, 4 260 17 Milk sugar-albumin mix 5 1.0 cornstarch, 0.1CaCO;, 0.1 19. Dried Torula yeast 5 38 KzHPOi. 20- National YeastAutolysate... 5 16 2l Soy enzyme hydrolysate 5 3 a K11, K12 and K13 areall stainless steel 100 gallon lermentors, which 22 Wheat protein acidhydrolysate 5 12 were generally charged with 65 gallons of medium; K10is a 750 gallon ron Mills). carbon steel termentor (500 gal. charge) andK4 was a 4,200 gal. carbon 23 Homogenized condensed fish 5 27 50 steeliermentor (3,200 gal. charge). 24- Protopeptone #366 (Wilson Labs)- 5 18Proflo is a detatted cottonseed product of Traders Oil Mill 00., Fort 25Cornsteep solids 5 26 Worth, Texas.

TABLE 7.EFFECT OF NUTRIENT VARIATIONS ON ANTI- TABLE 10 PRODUCTION 0ANTIBIOTIC BY S R gig'lglgs YIELD BY STREPTOMYCES X-5108 IN SHAKE MYCESX45103 0N SYN HETIC MEDIA Antibiotic Antibiotic Age in potency inE P inComposition of medium, in percent days units/ml. mont n E units numberComposition of medium in percent days per m1. L0 Na glutamate, 1 dexmn,01 11 11 20 5 MgSO4 s 7. 6 1 1 meat meal digest, l dextrln 4 40 tum 1 1d t K HPO 2 1 iiarnezitiation grain residue (BY-100), 4 67 1 gfi f iifex 2 5 ll. 5 T BBL Czapek-Dox broth 5 4.4 3 1 defatted cottonseed flour1 4 52 1.0 Arginine, 1.9 dextl'in, 9.1 111111 0., 9.1

dextrm- CBCOa, 9.05 M soi 0 0 lfish scraps 4 40 1.9 dl-aspartic acid,1.0 dextrin, 0.1 KzHPOi, 5- 1 corn distiller's qried grains with 6 1380,1 C8603, M5 so 6 0 g solubles (H. Walker), 1 dextrln- 0.1 Naglutamate, 0.6 (NH4)2HPO4, 1 glucose, butno C800: 4 19 1 dextrin, 0.1KzHPOi, 0.1 CaCOa, 9.1 K01, (d) but no CtlCO; 01 K2HPO4 4 34 005 M so 56 6 but only 11-25 Pro 4 12 9.33 nnezsoi, ucose, 0.1 Na citrate, 0.95but 4 47 Na acetate, 0.5 NaCl, 0.925 MgSOt-7H20, 0.91 1 Pl'OfiO, 1glucose. 6 21 K HPO1 0.01 KHzPol 0.3 CBCOa, 0.901 1 Profio, 1 starch- 621 1111180141110, 0 904 21150-111110, 1.6Xl0' l ProfiO, 1 glyeer 4 35KZCHOT 7 1 Profio, 1 brown u 4 14 1 PM), 1 maltose 4 1 Staleys sto-MiiioB. 1 f 11801050: 2 3% sucrose, 9.3% NaN01, 0.1 Kglll01,9.05 M soi, 9.95KC], 0.001 1 7 1 1 1 rosoi, as sold by Baltimore BiologicalLaboratories.

TABLE 11.-IN VI'IRO ANTIMICROBIAL SPECTRA OF ANTIBIOTIC X-5108 Diameterof inhibition zones 1 in mm.

Crude sodium salt Pure sodium salt of antibiotic of antiblotlc Testorganisms X-5108-1 rug/ml. X-5108-1 mg./m1.

Escherichia coli 17.3 (18) 18.0 (18.5). Paecilomyces vario (0).-- 0 (0).Mycobacterium phlei 19 20.2 Bacillus simplex 3113 (20) 40.3 (20)Pseudomonas aeruginosa 14.0 (125).-.. 16.0 (13). Aerobacler aerogenes12.0 (145).-.. 14.3 (15.5) Streptomyces cellulosae 31.3 (21)--. 33.0(21) Sarcim luleu 27.7 (21.5) 29.7 (22) Bacillus E-.- 36.3 (31) 37.2"(32W. Bacillus subtt 16.0 (17 17.0 (17 Serratia marcescens. 14.7 02).15.7 22.5). Candida albictms Trace (0) Trace (0). Peuicillium digilalum0 0. Saccharomyces cerevisiae 0(0) 0 (0). Staphylococcus aureus. 14.5(13).. 15.8 (14). Bodenhcimers bacillus. 20. B (18).. 21.7" (18).Proteus vulgaris 16.3 (20) 16.7 (20) 1 Where there are double entries,with entry in the upper right corner, it is indicative of the presenceof a secondary, hazier zone of inhibition. The abbreviations of es andens stand for clear and sharp, or for clear not sharp zone edges,respectively.

2 At 0.001 mg./ml. concentration.

a At 0.1 mgJml. concentration; for Bacillus E at 0.01 mg./m1., zone sizewas 22 mm. for J306.

I claim:

1. A composition for enhancing the feed efficiency and stimulating thegrowth of poultry which comprises antibiotic X-5108, a yellow amorphoussubstance characterized as follows:

(a) analysis: carbon, 63.63%; hydrogen, 7.81%; ni-

trogen, 3.48%; oxygen, 25.08% (by difference);

(b) soluble in methanol, ethanol, 1- and 2-propanol,

tert. butyl alcohol, ethyl acetate, amyl acetate, butyl acetate andchloroform;

(c) a characteristic infrared absorption spectrum as shown inaccompanying FIG. 1;

(d) a characteristic nuclear magnetic resonance spectrum as shown inaccompanying FIG. 4; or a pharmaceutically acceptable salt thereof, anda nontoxic inert ingredient, wherein antibiotic X-5108 or a salt thereofis present in a ratio by weight of from about 0.0001 part to about 0.01part per 100 parts of inert ingredient.

2. The composition of claim 1 wherein said non-toxic inert ingredient isa high energy nutrient feed for poultry containing a carbohydrate sourceand a protein source.

3. The composition of claim 1 wherein an alkali metal salt of antibioticX-S 108 is present.

4. A composition for enhancing the feed efficiency and stimulating thegrowth of poultrywhich comprises a con- 18 centrated pro-mixture ofantibiotic X-5108, said antibiotic being a yellow amorphous substancecharacterized as follows:

(a) analysis: carbon, 63.63%; hydrogen, 7.71%; nitrogen, 3.48%; oxygen,25.08% (by difference);

(b) soluble in methanol, ethanol, 1- and 2-propanol, tert. butylalcohol, ethyl acetate, amyl acetate, butyl acetate and chloroform;

(c) a characteristic infrared absorption spectrum as shown inaccompanying FIG. 1;

(d) a characteristic nuclear magnetic resonance spectrum as shown inaccompanying FIG. 4;

or a pharmaceutically acceptable salt thereof, and an inert, non-toxicingredient wherein said pre-mixture upon addition to a coventionalpoultry feed containing a protein source and a carbohydrate source,produces a feed containing per parts by weight of feed from about 00001to about 0.01 part by weight of antibiotic X5108 or a pharmaceuticallyacceptable salt thereof.

5. A method for stimulating the growth and enhancing the feed efiiciencyof poultry which comprises orally administering to poultry as theirregular feed requirement a composition containing antibiotic X-5108, ayellow amorphous substance characterized as follows:

(a) analysis: carbon, 63.63%; hydrogen, 7.81%; ni-

trogen, 3.48%; oxygen, 25.08% (by difference);

(b) soluble in methanol, ethanol, 1- and 2-propanol, tert. butylalcohol, ethyl acetate, amyl acetate, butyl acetate and chloroform;

(c) a characteristic infrared absorption spectrum as shown inaccompanying FIG. 1;

(d) a characteristic nuclear magnetic resonance spectrum as shown inaccompanying FIG. 4;

or a pharmaceutically acceptable salt thereof, and a high energynutrient feed for poultry containing a carbohydrate source and a proteinsource, wherein said antibiotic X-5108 or said salt thereof is presentin a ratio by weight of from about 0.0001 part to about 0.01 part ofantibiotic X-5108 or a salt thereof per 100 parts feed.

References Cited Miller: The Pfizer Handbook of Microbial Metabolites,McGraw-Hill Book Co., Inc., New York, N.Y., 1961, p. 125.

JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. 424-l2l H050 UNHEDSTATES FATE @FFHQE errmeme r memo Patent No. a 6R7 w] Dated April 18,1972 lxwentorw) Julius Berger It 1e certified that error appears in theebove identified patent end that said Letters Patent ere herebycorrected d5 shown bels Column 1, line 12 "Styreptomyces" should beStreptomyces Column 1, line 68 "those prepared of Difco" should be thoseprepared by Dif'co Column 4, line 63 "by gel permeation cbromatograph"should be by gel permeation chromatography Column 9, line 38 "addeddiethylsodiomalonace reagent to pH 10;"

should be added diethylsodiomalonate reagent to pH 8,0;

Signed and sealed this 23rd day of January 1973.

(SEAL) Attes't:

EDWARD M. FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents.

